Proton transfer reactions of acyl-CoA derivatives catalyzed by the enzyme tholase and vinylacetyl-CoA isomerase will be investigated. The major long-range goal of the proposed research is to understand how enzymes effectively catalyze the enolization of these very weak carbon acids. Both thiolase and vinylacetyl-CoA isomerase are involved in the oxidation of fatty acids. Thiolase is additionally involved in the biosynthesis of isoprenoids, including cholesterol. A knowledge of the mechanism of catalysis of these enzymes will be important in the future design of selective metabolic inhibitors of these and other enzymes -- particularly, the class of selective mechansim based inhibitors know as "suicide inhibitors," many of which require proton abstration for activation. The immediate experimental goals for the thiolase system are to determine how acetyl-CoA (the substrate for the enzyme) inactivates thiolase in a time-dependent fashion, and to ioslate and study the proton transfer step by examining a chemically modified form of thiolase which we have shown can catalyze acetyl-CoA enolization in the absence of the overall thiolase reaction. The immediate experimental goals for the vinylacetyl-CoA isomerase system are to fully characterize the proton transfer reaction of this enzyme by determining how protons partition between substrate, product and solvent during catalysis of the overall chemical reaction and to determine if the enzyme reaction proceeds through a stable carbanion/enol intermediate of acyl-CoA. Potential alternate substrates and inhibitors in which certain mechanisms of catalysis cannot participate will also be studied.